• Journal of Microbiology and Biotechnology
• /
• 제22권8호
• /
• pp.1107-1112
• /
• 2012

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE whole-cell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.

• 한국수산과학회지
• /
• 제42권1호
• /
• pp.20-25
• /
• 2009

Thirty-eight Vibrio spp. were isolated from the sea waters harvested from the 22 stations located on the west coast of the Korean peninsula in September 2006. The isolates consisted of V. parahaemolyticus (n=21), V. alginolyticus (n= 16) and V. cholerae non-01 (n=1), among which 35 isolates displayed resistance against two of the tested antibiotics. Among the 38 isolates, 18 isolates exhibited multi-drug resistance against more than four 4 antibiotics. In particular, minimum inhibitory concentration $(MIC)_{50}$ and $MIC_{90}$ of ampicillin-resistant isolates were as high as $2,048{\mu}g{\cdot}mL^{-1}$ and $4,096{\mu}g{\cdot}mL^{-1}$ respectively. $\beta$-lactamase production was examined to analyze the ampicillin-resistance. Some Vibrio spp. isolates produced $\beta$-lactamase, however antibiotics resistance pattern and $\beta$-lactamase production were not clearly related to each other. A genetic relationship between resistance and gene expression was confirmed in the ampicillin-resistant isolates.

• Journal of Microbiology and Biotechnology
• /
• 제22권4호
• /
• pp.555-562
• /
• 2012

PCR was performed to analyze the ${\beta}$-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known ${\beta}$-lactamase genes. This prompted us to screen new ${\beta}$-lactamase genes. A novel ${\beta}$-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 ${\beta}$-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other ${\beta}$-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A ${\beta}$-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 ${\beta}$-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various ${\beta}$-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 ${\beta}$-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 ${\beta}$-lactamase gene, led to the assumption that the location of this new ${\beta}$-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 ${\beta}$-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 ${\beta}$-lactamase is a new and separate member of class A ${\beta}$-lactamases.

• 대한미생물학회지
• /
• 제34권6호
• /
• pp.513-518
• /
• 1999

A total of 113 Vibrio sp. strains were examined for plasmid content which were subjected to digestion with restriction enzymes. About the 55% Vibrio spp. have the plasmid more than one. Most of these plasmid various derivatives ranged from $2.4\;kb{\sim}23\;kb$, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between $70\;kb{\sim}100\;kb$). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH and CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 9 strains of V. parahaemolyticus and 1 strain of V. alginolyticus from clinical isolates and 1 strains of V. mimicus from environmental isolates. In the experiments of tdh gene detection, in all, 3 strains of V. parahaemolyticus from clinical isolates and 2 strains from environmental isolates could be successfully amplified in 400 bp by PCR. The PCR results were consistent with DNA hybridization tests. In the experiments of CT assay, in all, 3 strains of V. cholerae from clinical isolate and 1 strain of V. cholerae from environmental isolates were observed CT-producing. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.

• 한국수산과학회지
• /
• 제10권1호
• /
• pp.31-36
• /
• 1977

우리나라 충무연안의 해양 미생물의 월별 분포를 파악하고 이에 따라 효과적인 양식업 대책수립은 물론, 어패류로 인한 식중독 발생 예방 재료를 얻고자 1976년 4월부터 1977년 3월까지 매월 해수, 이토, 어류 및 패류등을 수집하여 조사한 결과를 요약하면 다음과 같다. 1. 해수, 이토, 어류 및 패류 732 시료에서 1,426 균주를 분리 하였다. 분리된 균종은 Pseudomonas fluorescens가 450균주, Achromobacter liquifacience 422 균주 Vibrio Parahaemolyticus가 72균주, V. alginolyticus 234균주, Proteus vulgaris가 248 균주였다. 2. V. parohaemolyticus는 732 시료중 72 균주로서 $9.84\%$ 였으며 이들중에도 해수, 이토에서 $52\%$이상을 차지하였다. 또 넓게 분포되어 있음을 알았다. 3. 양식장(뗏목)의 이토를 채취 했을 때 이토속에서 많은 Gas가 발생함을 알았고 이것은 대부분 패류의 분비물 퇴적으로 추정되었다.. (부패가 일어남) 또한 이러할 곳은 용존산소량이 적을뿐 아니라, 패류에 산소가 부족되면 신진대사가 잘 안되므로 비만도(肥滿度)가 저하한다. 이러한 해역에서 폐사된 패류를 많이 볼 수 있었다. 4. 조사지역별 분포를 보면 Station 10, 9, 8, 11, 1의 순이였다. (Fig. 1)특히 St. 10은 여객선이 많이 출입하는 충무항이며 Station 9는 주위 매축공사로 인하여 분포율이 컸고 St. 8은 분뇨처리 탱크가 가까이 있기 때문에 영향이 크다고 느껴진다. 본 연구는 1976년도 문교부 학술연구 조성비로 이루어졌다. 통영수전 한학수 교장님과 문교부에 감사를 드리며 본 실험을 도운 이정태 조교님, 재료 학명등에 협조하여 주신 김무상 교수님, 가공과 신영호, 강숙희양께 사의를 표한다.

• Fisheries and Aquatic Sciences
• /
• 제5권3호
• /
• pp.178-182
• /
• 2002

The authors identified 68 Vibrio strains from Gwangan beach seawater from June to October in 2001. We identified them as 19 strains of Vibrio alginolyticus, 15 strains of V. vulnificus, 15 strains of V. parahaemolyticus, 11 strains of V. cholerae non O1, 7 strains of V. fluvialis and just one strain of V. hollisae. They showed their typical biochemical characteristics by API 20E kit (bioMerieux), respectively. It was examined whether their cultural supernatants had enzymatic activities such as hemolysin, protease or urease. The 46 strains showed hemolytic activities and/or protease activities. But we could not find any strain which had urease activity. All isolates of V. cholerae non O1 showed $\beta$ hemolysis. The others showed $\alpha$ hemolysis or did not show clear zones on sheep blood agar plates. These results of Kanagawa phenomenon were not always correspondant with hemolytic activities of cultural supernatants at late log phase. Some strains had higher hemolytic activities despite of showing protease activities on skim milk agar plates and in litmus milk media. On the other hand, some strains showed protease activities but did not show hemolytic activities. Therefore we could guess that there were the relationships between hemolysins and proteases produced by pathogenic vibrios.

• 대한미생물학회지
• /
• 제34권5호
• /
• pp.423-433
• /
• 1999

A novel bacteriophage, designated as VPP97, that infects the strains of Vibiro parahaemolyticus (hallophilic, Gram-negative bacterium) isolated most commonly from marine environments, has been discovered, and several of its properties have been determined. The plaques were clear and sized $0.6{\sim}1.0\;mm$ in diameter. The virion forms a single band on 70% sucrose gradient and ${\rho}1.50$ CsCl gradient by sucrose gradient centrifugation and CsCl gradient centrifugation respectively. It has a hexagonal head and a relatively long tail, as shown by electron microscopy. Vibrio alginolyticus, Vibrio fluvialis and Vibrio furnissii were also sensitive to this phage. It was almost totally inactivated at $70^{\circ}C$ and at pH below 5 or over 10. The nucleic acid of VPP97 is composed of DNA. The VPP97 had 9 specific structural proteins sized between 21.5 kDa and 97.4 kDa on SDS-PAGE. When V. parahaemolyticus cultures were treated with either phage VPP97 or one of the several antibiotics for 2 hours, the viable number of V. parahaemolyticus treated with the phage VPP97 is lower than that treated with chloramphenicol, erythromycin or penicillin, but not lower than that treated with tetracycline. Mice that have responded to the phage treatment revealed the lower numbers of V. parahaemolyticus in small intestine and less damage on small intestine compared to the untreated mice. Therefore, we suggest that the phage treatment appears effective to the infection by V. parahaemolyticus.

• 한국식품위생안전성학회지
• /
• 제30권3호
• /
• pp.281-288
• /
• 2015

Vibrio 속에 속한 세균에 의한 식중독은 오염된 해산물 식품의 섭취로 인하여 빈번하게 발생하고 있다. 그러므로 해산물을 날것으로 섭취하는 한국인의 특성을 고려할 때, 빠르고 정확한 Vibrio 검출기술은 식품위생 및 공중보건의 측면에서 매우 중요하다. 이와 관련하여 본 연구에서는 전통적인 배지를 이용한 동정방법의 단점을 보완할 수 있는 생화학적 방법인 Vitek 2 system방법과 분자생물학적 방법인 species-specific PCR 방법을 사용하여 얻은 동정결과를 비교 평가하고자 하였다. 본 연구에서는 5개의 Vibrio 표준균주와 16S rRNA gene sequencing 결과에 의하여 Vibrio 속으로 확인된 24개의 분리균주가 이용되었다. Vitek 2 system방법을 이용한 경우, 이와 같이 본 연구에 사용된 29개 균주 중 Vibrio 표준균주 2개(2/5, 40%), 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 15개(15/24, 62.5%) 등의 총 17개 균주(17/29, 58.6%)가 Vibrio 종으로 동정되었다. 그리고 species-specific PCR방법을 이용한 경우, 위의 29개 균주 중 Vibrio 표준균주 5개(5/5, 100%), 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 16개(16/24, 66.7%) 등의 총 21개 균주(21/29, 72.5%)가 Vibrio 종으로 동정되었다. Vitek 2 system방법과 species-specific PCR방법을 이용하여 동정된 결과를 비교하였을 때 표준균주 중 V. parahaemolyticus, V. mimicus 등의 2개(2/5, 40%), 새우분리균주 24개 중에서 16S rRNA gene sequencing 결과 Vibrio 속으로 확인된 분리균주 7개 (7/24, 29.2%) 등의 총 9개(9/29, 31%) 균주들에 대한 동정결과만이 일치하였다.

• 한국수산과학회지
• /
• 제30권3호
• /
• pp.361-366
• /
• 1997

금강 하구의 물에서 분리된 sucrose 분해성 Vibrio 속 중에 용혈독소를 생산하는 균주의 독성을 시험한 결과와 기존에 알려진 12종의 병원성 Vibrio 녹과 비교한 결과는 다음과 같다. 1. 분리된 장소의 환경조건은 염도가 $4.7\%_{\circ}$, pH가 7.6, 수온이 $24^{\circ}C$ 및 conductivity가 $7800{\mu}MHOS$이었다. 2. 생리, 생화학적 특성과 염분 요구도를 비교한 결과 sucrose를 분해하는 병원성 Vibrio인 V. alginolyticus, V. cholerae, V. cincinnatiensis, V. fluvialis, V. furnissii 및 V. metschnikouii와는 확실히 구별되었다. 3. 생육가능한 염도는 $0.5\~7.5\%_{\circ}$이었으며 생육가능한 pH는 $4.5\~9.5$이었다. 4. TCBS 평판한천배지에서 균의 집락은 전형적인 황색집락이었으며, 전자현미경에 나타난 균의 형태은 콤마상 간균이었다. 5. 새앙쥐에 대한 치사독성이 확실하였으며, 사람과 면양 적혈구에 대한 용혈성이 확인되었고 rat 피부혈관 투과성 항진작용이 양성이었다. 이상의 결과로 이 균은 병원성이 확인되었고 기존에 알려지지 않은 Vibrio 속으로 확인되어 이 균을 Vibrio sp. D5로 명명하였다.

• Fisheries and Aquatic Sciences
• /
• 제22권2호
• /
• pp.5.1-5.9
• /
• 2019

Background: Live fish import may lead to the unintended introduction of pathogens. We examined the monthly distribution of microbial pathogens in ornamental finfish imported into South Korea over a 6-month period. Results: Vibrio alginolyticus was detected in one lemon damsel in June and July; V. vulnificus was detected in one lemon damsel, one caerulean damsel, and one pearl-spot chromis and one ocellaris clownfish in July, April, and May, respectively; Photobacterium damselae was detected in one ocellaris clownfish and one caerulean damsel in June and July, respectively; V. anguillarum was detected in one pearl-spot chromis in February; V. harveyi was detected in one ocellaris clownfish and two mandarin fish in February and April, respectively; Yersinia ruckeri was detected in a pearlscale goldfish group in June and July and in two colored carp groups in July; and Lactococcus garvieae was detected in a lemon damsel group and a sutchi catfish group in July and May, respectively. European catfish virus, the only viral pathogen detected, was found in two sutchi catfish groups in May. Conclusion: This study is the first to identify pathogenic species and the presence or absence of pathogens (non-quarantine diseases) in imported ornamental finfish. These results demonstrate that various pathogens with the potential to harm indigenous fish populations can accompany ornamental finfish imported into South Korea.